1. Field of the Invention
The present disclosure relates to the generation of neural cells, most preferably human dopaminergic neurons, derived from embryonic-like stem cells isolated from corneal limbus tissue. In particularly preferred embodiments, the dopaminergic neurons disclosed herein are used for cell-replacement therapy in various neurodegenerative diseases including, but not limited to, Parkinson's disease. Methods for generating and isolating neural precursor cells and methods for their use are also disclosed.
2. Description of Related Art
I. Stem Cells
In early development, the ultimate source of all tissues in a mammalian embryo or fetus is the stem cell population. In the embryonic stage, embryonic stem cells (ES cells) are totipotent and therefore capable of developing into all the cells of a complete organism. Cellular development occurs through several phases, including cellular proliferation, lineage commitment, and lineage progression, resulting in the formation of differentiated cell types. There are three main lineages that are derived from embryonic germ layers: ectoderm, mesoderm, and endoderm. The ectoderm germ layer forms the epidermis of the skin, sense organs, nervous system, and spinal cord. The mesoderm germ layer forms smooth muscle, connective tissues, blood vessels, heart, blood cells, bone marrow, reproductive organs, the excretory system, striated muscles, and skeletal muscles. Finally, the endoderm germ layer forms epithelial linings of the respiratory and gastrointestinal tracts, including the pharynx, esophagus, stomach, intestine, and other associated organs. ES cells are often referred to as pluripotent stem cells because they are not fixed in their developmental potentialities and can differentiate into many different cell types in vitro.
Because ES cells are capable of becoming almost all of the specialized cells of the body, they have the potential to generate replacement cells for a broad array of tissues and organs such as heart, pancreas, nervous tissue, muscle, cartilage, and the like. ES cells can be derived from the inner cell mass (ICM) of a blastocyst, which is a stage of embryo development that occurs prior to implantation. ES cells derived from the ICM can be cultured in vitro and, under the appropriate conditions, proliferate indefinitely. ES cell lines have been successfully established for a number of species, including mouse (Evans et al., 1981, Nature 292:154-156), rat (Iannaccone et al., 1994, Dev. Biol., 163:288-292), rabbit (Giles et al., 1993, Mol. Reprod. Dev. 36:130-138; Graves et al., 1993, Mol. Reprod. Dev. 36:424-433), hamster (Doetschman et al., 1988, Dev. Biol. 127:224-227), primate (U.S. Pat. No. 5,843,780), and human (Thomson et al., 1998, Science 282:1145-1147; Reubinoff et al., 2000, Nature Biotech. 18:399-403).
The isolation of human ES cells offers the promise of a remarkable array of novel therapeutics. Biologic therapies derived from such cells, including tissue regeneration and repair, as well as targeted delivery of genetic material, are expected to be effective in the treatment of a wide range of medical conditions. Despite the enormous potential of these materials, however, serious ethical issues related to the use of human pluripotent stem cells derived from human embryos or from fetal tissue obtained from terminated pregnancies make stem cell research and treatments involving stem cells controversial. In addition, there are technical issues associated with the use of ES cells. For example, tissues or cells derived from ES cells are not ideal for use in medical treatments because ES cells will generally not be derived from the patient who will ultimately receive the treatment. It is well-known that the use of autologous tissues is preferred for transplant therapies in order to avoid tissue rejection problems, which may prove difficult in the area of stem-cell-based therapies.
The problems associated with human ES cells led many researchers to turn their attention to adult tissues as a possible source of undifferentiated stem cells with properties similar to those of ES cells or germ cells derived from fetal tissue. It is known that after birth and throughout adulthood, a small number of specialized stem cells are retained in an organism for the replacement of cells and the regeneration of tissues. These adult stem cells (also referred to as “tissue-specific stem cells”) have been found in very small numbers in various tissues of the adult body, including bone marrow, (Weissman, 2000, Science 287:1442-1446), neural tissue (Gage, 2000, Science 287:1433-1438), gastrointestinal tissue (Potten, 1998, Phil. Trans. R. Soc. Lond. B. 353:821-830), epidermal tissue (Watt, 1997, Phil. Trans. R. Soc. Lond. B. 353:831), hepatic tissue (Alison and Sarraf, 1998, J. Hepatol. 29:678-683), and mesenchymal tissue. (Pittenger et al., 1999, Science 284:143-147).
While some potential sources of adult stem cells have been identified, to date, adult stem cells have not been found to be an adequate replacement for ES cells. First, adult stem cells can be difficult to isolate because they are usually present only in minute quantities in tissues that are often not easily accessible, and their numbers appear to decrease with age. Second, adult stem cells appear to be a less desirable source of cultured tissue than ES cells because they have a shorter life span and less capacity for self-renewal. Third, adult stem cells are believed to be tissue-specific and not pluripotent, generally capable of giving rise only to new cells of a few types closely related to their tissue of origin.
One particularly notable difference between ES cells and adult stem cells is that ES cells in suspension culture are capable of forming aggregates of cells known as embryoid bodies. These embryoid bodies usually contain germ cells of all three lineages that differentiate into various lineage-committed tissues. Therefore, embryoid bodies can be useful in the preparation of different types of differentiated cells in culture. To date, no other isolated adult stem cell lines have been reported that are capable of forming structures similar to embryoid bodies in culture.
Recently, however, it has been suggested that some adult stem cells have the capacity to be pluripotent. The most fully characterized are hematopoietic stem cells known as bone marrow stromal cells or mesenchymal stem cells (Jiang et al., 2002, Nature 418:41-48). These are the first adult stem cells found to have pluripotent properties. Pluripotent adult stem cells have also been isolated from liver (U.S. Publ. No. 2003/0186439), mouse inner ear (Li and Heller, 2003, Nat. Med. 9:1293-1299), and amniotic fluid (Prusa et al., 2003, Hum. Reprod. 18:1489-1493). Recently, pluripotent adult stem cells have been described in many tissues such as skeletal muscle, brain, and intestinal epithelium (Howell et al., 2003, Ann. N.Y. Acad. Sci. 996:158-173.). Still, while these papers report isolated or identified adult stem cells that are pluripotent, these “pluripotent” adult stem cells, unlike ES cells, differentiate into only a few lineages. In addition, none of the isolated adult stem cells reported to date appear to be capable of forming embryoid-like bodies in culture in a manner similar to ES cells.
Similar to the other sources of adult stem cells referenced above, it is known that adult stem cells are present in the corneoscleral limbus of the eye. These cells participate in the dynamic equilibrium of the corneal surface and replace superficial epithelial cells that are shed and sloughed off during eye-blinking. Severe damage to the limbal stem cells from chemical or thermal burns, contact lenses, severe microbial infection, multiple surgical procedures, cryotherapy, or diseases such as Steven-Johnson syndrome or ocular cicatrical pemphigoid can lead to destruction of limbal stem cells and limbal stem cell deficiency, which can lead to an abnormal corneal surface, photophobia, and reduced vision (Anderson et al., 2001, Br. J. Opthalmol. 85:567-575). This damage cannot be repaired without the reintroduction of a source of limbal stem cells (Tseng et al., 1998, Arch. Ophthalmol. 116:431-41; Tsai et al, 2000, N. Engl. J. Med. 343:86-93; Henderson et al., 2001, Br. J. Ophthalmol. 85:604-609).
Experiments conducted in the 1980's first indicated the existence of limbal stem cells in the corneal epithelium (Schermer et al., 1986, J. Cell Biol. 103:49-62; Cotsarelis et al., 1989, Cell 57:201-209). Although it was later suggested that the transcription factor p63 was a specific marker for human corneal stem cells, this marker is also expressed in other epithelial cells such as skin, and therefore is not specific to corneal stem cells. In addition, although p63 expression has been shown to be principally limited to the basal limbal region in human corneas (Moore et al., 2002, DNA Cell Biol. 21:443-51), expression of this transcription factor in mice was maximal in paracentral cornea tissue rather than limbus (Moore et al., 2002, DNA Cell Biol. 21:443-451). Therefore, there is currently no known definitive stem cell marker for limbal epithelial stem cells.
It would be desirable to identify a source of adult stem cells that are capable of self-renewal in culture and are pluripotent and ES cell-like in their ability to differentiate into cells of all three major lineages: ectoderm, mesoderm, and endoderm. Further, it would be desirable to isolate and culture these adult stem cells, and to induce them to differentiate into various cell types, such as, for example, neuronal cells.
II. Neurodegenerative Disorders
Neurodegenerative disorders and neuronal diseases such as Parkinson's disease, Alzheimer's disease, and schizophrenia are destructive diseases that are becoming ever more prominent in our society. Many of these neurological disorders are associated with dopaminergic neurons. Dopaminergic neurons reside in the ventral and ventro-lateral aspects of the midbrain, and control postural reflexes, movement, and reward-associated behaviors. These neurons innervate multiple structures in the forebrain, and their degeneration or abnormal function is associated with Parkinson's disease, schizophrenia, and drug addiction (Hynes et al., 1995, Cell 80:95-101).
Parkinson's disease is a progressive neurological disorder caused by the degeneration of neurons in the region of the brain that controls movements. This degeneration creates a shortage of dopamine, causing the movement impairments that characterize the disease. Pathological studies indicate that loss of dopaminergic neurons in the substantia nigra contributes to Parkinson's disease (Ungerstedt, 1971, U. Acta Physiol. Scand. Suppl. 367:95-121; Yirek and Sladek, 1990, Annu. Rev. Neurosci. 13:415-440). In parkinsonism, changes in the status of dopaminergic receptors may be dependent on the stage of progression of the disease. The hallmark of parkinsonism is a severe reduction of dopamine in all components of the basal ganglia (Hornykiewicz, 1988, Mt. Sinai J. Med. 55:11-20). When dopamine is depleted, various other areas in the brain such as the thalamus, globus pallidus, and the subthalamic nucleus start to malfunction. Since these areas send signals to other parts of the brain, malfunctions in these small areas can lead to widespread brain dysfunction.
The prevalence of Parkinson's disease varies widely from 82 per 100,000 in Japan and 108 per 100,000 in UK, to nearly 1% (approximately 1 million) of the population in North America. In India, the prevalence rate of Parkinson's disease is 14 per 100,000 in North India, 27 per 100,000 in South India, 16 per 100,000 in East India, and 363 per 100,000 for the Parsi community in Western India. While Parkinson's disease is currently considered incurable, a variety of medications are available that provide symptomatic relief from Parkinson's disease, including Levodopa, Bromocriptine, pergolide, selegiline, anticholinergic, and amantadine. Although these drugs may provide relief from the symptoms of Parkinson's disease, they often have significant side effects. Moreover, these drugs neither cure the disease nor slow down the progressive loss of neurons, and only relieve the symptoms, with the beneficial effects often wearing off with time.
These unsatisfactory outcomes have promoted the development of other strategies for treating this disease, such as dopa-receptor agonist therapy and surgical approaches that include pallidotomy, deep brain stimulation (DBS) of the globus pallidus, and attempts to interrupt network abnormalities by destroying overactive brain areas or placing DBS electrodes to quiet these areas. Although these and other surgeries have produced some beneficial results in patients with Parkinson's disease, the long-term effects of such surgeries are not yet known. These treatments also have certain limitations and side effects. Because of the limitations of current treatments, several new strategies are being pursued to develop novel therapies for patients with Parkinson's disease. These techniques range from the use of dopaminotrophic factors (Takayama, et al., 1995, Nature Med. 1:53-58) and viral vectors (Choi-Lundberg et al., 1997, Science 275:838-841) to the transplantation of primary xenogeneic tissue (Deacon et al, 1997, Nature Med. 3:350-353). In addition, gene therapy is being pursued as a therapeutic strategy for this disease (Zurn et al., 2001, Brain Res Rev. 36:222-229; Date et al., 2001, Cell Transplant 10:397-401; Akerud et al., 2001, J. Neurosci. 21:8108-8118).
Cell implantation is another therapeutic strategy that offers the hope of replacing nerve cells lost in Parkinson's disease, as well as other neurodegenerative disorders and neuronal diseases. Clinical trials with fetal tissue transplantation, still underway, have demonstrated methods for implanting cells into the brain and the viability of this concept, as well as produced promising results for at least some patients (Freed et al., 2001, N. Engl. J. Med. 344:710-719; Winkler et al., 2000, Prog. Brain Res. 127:233-265). In one study, transplantation of dopaminergic neurons into the substantia nigra of a patient with Parkinson's disease was described as therapeutically effective, but symptomatic relief was incomplete (Lindvall, 1997, Neuroreport. 8(14):iii-x), indicating that transplantation of dopaminergic neurons alone may not be sufficient to cure Parkinson's disease.
Recently, a renewable source of neural stem cells was discovered in the adult human brain. Neural stem cells with the capacity to renew themselves and form all cell types of the brain offer a potentially unlimited supply of dopamine-producing brain cells, thus promising an entirely new therapeutic approach to neurodegenerative disorders and neuronal diseases (Eriksson et al., 1998, Nature Medicine 4:1313-1317). It has been reported that cultures of neural stem cells derived from the embryonic human forebrain can be expanded up to ten million fold in vitro. These adult neural stem cells were transplanted into adult rats that serve as a well-characterized model of Parkinson's disease. The transplanted cells survived for up to a year after transplantation, differentiated into neurons, and improved motor disorders in some of the experimental animals (Svendsen et aL., 1997, Exp. Neurol. 148:135-146). Unfortunately, adult neural stem cells have a limited life span in tissue culture (Kukekov et al., 1999, Exp. Neurol. 156:333-344).
One viable alternative source of dopaminergic neurons, and other neurons that may be used to treat various neurodegenerative disorders and neuronal diseases, are pluripotent ES cells. Studies have shown that ES cells can be differentiated into neural progenitor cells (Zhang et al., 2001, Nature Biotech. 19:1129-33; WO 01/88104; U.S. Pat. No. 6,833,269; U.S. Ser. Nos. 09/888,309, 10/157,288; WO 03/000868; each specifically incorporated herein by reference). These cells can then be further differentiated into dopaminergic neurons (Rolletschek et al., 2001, Mech. Dev. 105:93-104, incorporated herein by reference). An initial step in the differentiation of ES cells can be the formation of embryoid bodies; for example, 1 mM of retinoic acid promotes neural differentiation into embryoid bodies (Bain et al., 1995, Dev. Biol. 168:342-357, incorporated herein by reference). While retinoic acid can be used to generate neural cells, retinoic acid is a strong teratogen.
Several reports have been published on the differentiation of ES cells into dopaminergic neurons by using stromal cell inducing activity (SIDA) (Kawasaki et al., 2000, Neuron 28:1-20), by expressing nuclear receptor related-l gene (Nurr-1) (Kim et al., 2002, Nature 418:50-56), or by transplanting undifferentiated ES cells directly into the mouse model (Bjorklund et al., 2002, Proc. Natl Acad. Sci. 99:2344-2349). Lee et al. reported a method for differentiating ES cells into neural progenitor cells and into dopaminergic and serotonergic neurons in vitro (Lee et al., 2000, Nat. Biotechnol. 18:675-79). All of these experiments, however, were carried out using mouse ES cells, and the differentiation protocols yielded dopaminergic neurons ranging from 5-50%. About 20% of the mouse ES cells developed into dopaminergic neurons in the study by Lee et al. (WO 01/83715) and 5-50% in the study by Studer et al. (WO 02/086073). While dopaminergic neurons have also been differentiated from human ES cells, yields of only 5-7% of dopaminergic neurons, as a percentage of total cells in the population, have been obtained (WO 03/000868).
Parkinson's disease is thought to be a particularly suitable clinical target for a cell transplant therapeutic strategy since it is characterized by the selective and gradual loss of dopaminergic neurons in the substantia nigra of the midbrain. The loss of dopamine-producing neurons within this specific brain site leads to abnormal firing of nerve cells that results in patients being unable to control or direct their movements. But one challenge of this approach is that large numbers of dopaminergic neurons are required for cell replacement therapy. Transplantation of dopaminergic neurons is a clinically promising experimental treatment for advanced stage Parkinson's disease. Cell transplantation therapy has been performed on more than 200 patients worldwide (Olanow et al., 1996, Trends Neurosci. 19:102-109). Clinical improvement has been confirmed (Olanow et al., supra and Wenning et al., 1997, Ann. Neurol. 42:95-107), and was correlated to good graft survival and innervation of the host striatum (Kordower et al., 1995, N. Engl. J. Med. 332:1118-1124). Unfortunately, fetal nigral transplantation therapy generally requires human fetal tissue from at least 3-5 embryos to obtain a clinically significant improvement in the patient. This poses an enormous logistical and ethical dilemma. Thus, alternative sources for dopaminergic neurons are being investigated.
For example, dopaminergic neurons have been generated from CNS precursor cells (WO00/005343; and Studer et al., 1998, Nature Neurosci. 1:290-295.). These precursor-derived neurons are functional in vitro and in vivo and restore behavioral deficits in a rat model of Parkinson's disease. Even though the primary mesencephalic CNS stem cell culture can provide differentiated dopaminergic neurons suitable for use in cell therapy for Parkinson's disease, the cell number provided by this method is limited. The percentage of differentiated dopaminergic neurons obtained from expanded mesencephalic precursors decreases as the cells are expanded more than about 10-100 fold. While mesencephalic precursors can generate about 10% to 15% dopaminergic neurons (out of total cell number) after 10-100 fold expansion, when the precursors are expanded 1000 fold, that number drops to only about 1%.
Efficient generation of dopaminergic neurons in culture is of particular interest in view of the therapeutic promise of cell therapy in Parkinson's disease. Because therapies currently available for treating neurological and neurodegenerative diseases are extremely limited, there is great interest in developing alternative therapies. While ES cells can provide an excellent source of dopaminergic neurons, their use is controversial due to ethical issues. Thus, it is desirable to identify an alternate source from which a high yield of clinically acceptable dopaminergic neurons for human clinical application can be generated. The present disclosure provides a method for derivation of dopaminergic neurons from a novel source of pluripotent stem cells isolated from the adult human eye. The therapeutic potential of these differentiated cells for neurodegenerative diseases, such as Parkinson's Disease, is examined.